Arachidonic acid (20:4) is the precursor of an array of potent oxidation products (eicosanoids) whose role in inflammation and hypersensitivity reactions is widely recognized. In neutrophils (PMN), various eicosanoids elicit chemotaxis, aggregation, and degranulation responses. Upon stimulation, the 20:4 is released from membrane phospholipids and converted to bioactive products. The release of 20:4 appears to be effected primarily by phospholipase A2 in PMN; however, many questions concerning the exact 20:4 donor and the nature of the specific enzyme(s) responsible for the release remain unanswered. In the studies proposed, these and other aspects of 20:4 metabolism will be explored. Recently our laboratory found that 45-50% of the choline-containing phosphoglycerides (PC) are alkyl-ether linked and that the alkyl-linked fraction is enriched in 20:4. Based on several recent findings, it is proposed that the 1-0-alkyl-2-arachidonoyl-GPC may act as an immediate precursor of both eicosanoids and platelet-activating factors; our studies suggest these mediators are formed and act in concert. the eicosanoids formed will be tested separately, and in combination, to evaluate their physiological and biochemical functions. Molecular species of phospholipids will be separated on the basis of chain length, the degree of unsaturation, and the nature of the sn-1 chain (alkyl, alk-1-enyl of acyl) by reverse-phase HPLC. By coupling labeling studies with this HPLC technique, the release of 20:4 from specific species and the fate of the lysophospholipid formed can be examined in detail; this information will be helpful in deducing the mechanism of 20:4 release. Stimuli will be compared to see if they promote release of 20:4 from different species, which then may be channeled into different products. The 20:4 content of PC derived by the phospholipid methylation pathway is to be measured and the release from this pool compared to that from other pools. The incorporation of 20:4 metabolites into the phospholipids is to be examined. We plan to use enucleated PMN to isolate events occurring in the plasma membrane and cytoplasm. The subcellular distribution of 20:4-containing phospholipids and of 20:4-metabolizing enzymes will be examined. The goals of these studies are to pinpoint the lipid donors and membrane sources of the 20:4 released upon stimulation and to elucidate the mechanism of release.